Figure 2.

COX2 downregulation is necessary for osteogenic differentiation of human SSCs. (A) The human skeletal stem cell (hSSC) lineage tree as defined by surface marker expression profiles with the hSSC at the apex and the downstream bone cartilage stroma progenitor (hBCSP) which gives rise to committed bone (hOP) and cartilage (hCPs) progenitor cells. (B) Microarray analysis showing COX-1 and COX-2 expression of freshly purified (uncultured) hSSCs from human fracture callus tissue of three different patients as well as their expression after two-week osteogenic differentiation from hSSCs. (C) Representative IHC images of COX2 staining in freshly purified hSSCs at different timepoints during in vitro osteogenesis. (D) Related quantitative PCR of COX-2 expression in the same experiment. n=12 independent replicates of hSSCs from four patients performed in triplicates. (E) Quantification of COX2 ICC staining by CTCF (corrected total cell fluorescence). n=20 independent replicates of hSSCs from two patients (n=10 each). Statistical testing between timepoints by one-way ANOVA with Tukey’s posthoc test. (F) Alizarin Red staining and quantification of hSSCs (with patient age; yo: years old) of lentivirally overexpressed COX2 or ZsGreen controls during osteogenic differentiation. n=6 independent replicates of hSSCs from three patients performed in duplicate. Statistical testing by unpaired Student’s t-test. All data shown as mean ± SEM. All results from at least two independent experiments.